Down-regulation of the Notch pathway mediated by a gamma-secretase inhibitor induces anti-tumour effects in mouse models of T-cell leukaemia.

BACKGROUND AND PURPOSE: gamma-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy. EXPERIMENTAL APPROACH: Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Abeta(40) and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours. KEY RESULTS: Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis. CONCLUSIONS AND IMPLICATIONS: Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.

p27 cytoplasmic localization is regulated by phosphorylation on Ser10 and is not a prerequisite for its proteolysis.

The activity of the cyclin-dependent kinase inhibitor p27 is controlled by its concentration and subcellular localization. However, the mechanisms that regulate its intracellular transport are poorly understood. Here we show that p27 is phosphorylated on Ser10 in vivo and that mutation of Ser10 to Ala inhibits p27 cytoplasmic relocalization in response to mitogenic stimulation. In contrast, a fraction of wild-type p27 and a p27(S10D)-phospho-mimetic mutant translocates to the cytoplasm in the presence of mitogens. G1 nuclear export of p27 and its Ser10 phosphorylation precede cyclin-dependent kinase 2 (Cdk2) activation and degradation of the bulk of p27. Interestingly, leptomycin B-mediated nuclear accumulation accelerates the turnover of endogenous p27; the p27(S10A) mutant, which is trapped in the nucleus, has a shorter half-life than wild-type p27 and the p27(S10D) mutant. In summary, p27 is efficiently degraded in the nucleus and phosphorylation of Ser10 is necessary for the nuclear to cytoplasmic redistribution of a fraction of p27 in response to mitogenic stimulation. This cytoplasmic localization may serve to decrease the abundance of p27 in the nucleus below a certain threshold required for activation of cyclin-Cdk2 complexes.

Ras-GAP SH3 domain binding protein (G3BP) is a modulator of USP10, a novel human ubiquitin specific protease.

Degradation of cellular proteins through ubiquitination is a fundamental strategy for regulating biological pathways. De-ubiquitination, i.e. the removal of ubiquitin from proteins and peptides to which ubiquitin is attached, is catalyzed by processing proteases known as de-ubiquitinating enzymes. We are studying the biology of a family of de-ubiquitinating enzymes, the mammalian ubiquitin-specific proteases (USPs), some of which appear to play a role in growth control. Given the fact that the modes of regulation of USPs and of their substrate specificity are poorly understood, we decided to attempt the identification of USP interacting proteins. Using the yeast two-hybrid system (2HS), we have isolated a cDNA clone whose product specifically interacts with USP10 but not with other USP baits tested. The isolated clone encodes a protein known to interact with the Ras-GTPase activating protein (G3BP). This interaction was further confirmed by performing a 2HS with G3BP, which led to the isolation of USP10 encoding cDNAs. We validated the interaction between the two proteins by performing in vitro binding assays and immunoprecipitations in human cells. G3BP does not appear to be a substrate of USP10; it rather inhibits the ability of USP10 to disassemble ubiquitin chains. The USP10/G3BP complex appears to co-immunoprecipitate with ubiquitinated species that could be substrates of USP10.

Human Cdc25 A inactivation in response to S phase inhibition and its role in preventing premature mitosis.

The Cdc25 A phosphatase is required for the G1-S transition of the cell cycle and is overexpressed in human cancers. We found that it is ubiquitylated and rapidly degraded by the proteasome and that its levels increase from G1 until mitosis. By treating cells with the DNA synthesis inhibitor hydroxyurea, Cdc25 A rapidly decreased in abundance, and this was accompanied by an increase in Cdk2 phosphotyrosine content and a decrease in Cdk2 kinase activity. Cdc25 A overexpression altered the ability of cells to arrest in the presence of hydroxyurea, and caused them to undergo premature chromosome condensation. Cdc25 A overexpression could render tumor cells less sensitive to DNA replication checkpoints, thereby contributing to their genomic instability.

Proteasome-dependent degradation of p27/kip1 in gliomas.

p27/kip1 regulates the G1-S transition of the cell cycle by inhibiting cyclin D-CDK4, cyclin E-CDK2, and cyclin A-CDK2. Modulation of p27 cellular abundance occurs mainly at post-translational level by the ubiquitin-proteasome proteolysis. Although rearrangements and mutations of p27/kip1 are extremely rare events, p27 levels are reduced and associated with a poor prognosis in many human carcinomas. In astrocytic tumors, p27 decreases with advancing anaplasia and is almost absent in glioblastomas. To verify whether the degradation of p27 protein was responsible for its reduced levels in malignant gliomas, p27 degradation activity was tested in 22 tissue extracts that represented high, low, and absent p27 protein levels. p27 protein expression was detected by immunohistochemistry and immunoblot analysis and comparable results between the 2 methods were obtained. Low or undetectable p27 degradation activity was found in samples that displayed high levels of p27, i.e. all 4 normal brain biopsies, and 4 out of 6 grade II astrocytomas. Enhanced degradation activity resulted in malignant gliomas with low or absent p27 protein levels. The proteasome inhibitor LLnL abolished p27 degradation, demonstrating that it occurs in a proteasome-dependent manner. These data suggest that proteasome degradation of p27 may be instrumental in the deregulation of the cell cycle and to the malignant transformation of gliomas.

Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation.

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor.

Down-regulation of p27 is associated with development of colorectal adenocarcinoma metastases.

The cyclin-dependent kinase inhibitor p27 is a negative regulator of the cell cycle and a potential tumor suppressor gene. Because we had previously demonstrated that loss of p27 protein is associated with aggressive behavior in colorectal adenocarcinomas, we used immunohistochemistry and in situ hybridization to evaluate the potential role of alterations in p27 expression in primary and metastatic colorectal adenocarcinomas. Parallel immunostaining was performed for Ki-67 and p53. We evaluated 13 cases of metachronous and 23 cases of synchronous primary and metastatic colorectal tumor pairs. In the synchronous subgroup (Stage IV tumors), 57% of the primary tumor and metastases pairs did not express p27 protein and the remainder were low expressors. In the metachronous subgroup, 54% of the primary tumors were low expressors and the remainder high expressors of p27 protein. There was a significant reduction in the expression of p27 in the metachronous metastases (mean positive cells: 14.5%) when compared to the corresponding primary tumors (mean positive cells: 41.8%), P = 0.0023. All the primary and metastatic tumors in the metachronous subgroup showed high levels of p27 mRNA expression. There was no association between loss of p27 and either Ki-67 count or p53 expression. Because p27 is known to be up-regulated when epithelial cells are grown in suspension, the down-regulation of p27 in circulating tumor cells may confer the ability to grow in an environment of altered extracellular matrix or intercellular adhesion properties, two situations which may facilitate metastases.

UBPY: a growth-regulated human ubiquitin isopeptidase.

The ubiquitin pathway has been implicated in the regulation of the abundance of proteins that control cell growth and proliferation. We have identified and characterized a novel human ubiquitin isopeptidase, UBPY, which both as a recombinant protein and upon immunoprecipitation from cell extracts is able to cleave linear or isopeptide-linked ubiquitin chains. UBPY accumulates upon growth stimulation of starved human fibroblasts, and its levels decrease in response to growth arrest induced by cell-cell contact. Inhibition of UBPY accumulation by antisense plasmid microinjection prevents fibroblasts from entering S-phase in response to serum stimulation. By increasing or decreasing the cellular abundance of UBPY or by overexpressing a catalytic site mutant, we detect substantial changes in the total pattern of protein ubiquitination, which correlate stringently with cell proliferation. Our results suggest that UBPY plays a role in regulating the overall function of the ubiquitin-proteasome pathway. Affecting the function of a specific UBP in vivo could provide novel tools for controlling mammalian cell proliferation.

11th European Cell Cycle Conference: April 23-26, 1997, Gardone Riviera (Italy) and Symposium on Cell Cycle Regulation (17th International Congress of Biochemistry and Molecular Biology): August 24-29, 1997, San Francisco, CA.

In the following pages I have summarized some of the findings presented at two recent 'cell cycle gatherings'. I have focused on those topics which in my opinion represent a substantial advancement in our understanding of the cell cycle regulatory pathways.

The cell cycle inhibitor p27 is an independent prognostic marker in small (T1a,b) invasive breast carcinomas.

Breast carcinomas < or = 1 cm in size (T1a,b) are being detected more frequently as a result of screening. Because traditional prognostic parameters are either lacking (tumor size) or rare (nodal metastases), a marker(s) is needed to identify the subset of patients who could benefit from adjuvant therapy. A retrospective series of 202 patients with stage T1a,b invasive breast carcinomas was evaluated. The clinicopathological features (age, histological grade, extensive in situ carcinoma, hormone receptor status, and nodal metastasis) as well as microvessel density and the expression of c-erb-B2, p53, MIB-1/Ki-67, and cdc25B were assessed. In addition, expression of the cell cycle inhibitor p27 was evaluated. Nineteen patients (18% of patients who had axillary dissection) had locoregional lymph node metastases. Forty-two % of them died of disease (median survival, 112 months), whereas mortality was 11% in node-negative patients (median survival, 168 months; P = 0.0055). Patients with low p27 expression had a median survival of 139 months (17% mortality) versus 174 months (9% mortality) in the group with high p27 expression (P = 0.0233). Lack of p27 was associated with poor prognosis when node-positive patients were excluded (P = 0.0252). Nodal status and low p27 were found to be the only independent prognostic parameters by both univariate and multivariate analysis, with relative risks of dying of disease of 4.9 (P = 0.001) and 3.4 (P = 0.0306), respectively. Assessment of p27, which yields prognostic information in node-negative patients, could be useful to identify patients with small, invasive breast carcinomas who might benefit from adjuvant therapy.

Interaction between Cdc37 and Cdk4 in human cells.

Using the yeast two-hybrid system we have identified novel potential Cdk4 interacting proteins. Here we described the interaction of Cdk4 with a human homologue of the yeast Drosophila CDC37 gene products. Cdc37 protein specifically interacts with Cdk4 and Cdk6, but not with Cdc2, Cdk2, Cdk3, Cdk5 and any of a number of cyclins tested. Cdc37 is not an inhibitor nor an activator of the Cdk4/cyclin D1 kinase, while it appears to facilitate complex assembly between Cdk4, and cyclin D1 in vitro. Cdc37 competes with p16 for binding to Cdk4, suggesting that p16 might exert part of its inhibitory function by affecting the formation of Cdk4/cyclin D1 complexes via Cdc37.

Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas.

The cell-cycle inhibitor p27 is a potential tumor suppressor, but its gene has never been found inactivated in human tumors. Because cell-cycle regulation of p27 cellular abundance occurs at the post-transcriptional level, we analyzed p27 protein expression and degradation in human colorectal carcinomas. Proteasome-mediated degradation activity of p27 was compared with its protein levels in a subset of tumor samples. We found that carcinomas with low or absent p27 protein displayed enhanced proteolytic activity specific for p27, suggesting that low p27 expression can result from increased proteasome-mediated degradation rather than altered gene expression. Patients whose tumors expressed p27 had a median survival of 151 months, whereas patients who lacked p27 (10%) had a median survival of 69 months. By multivariate analysis, p27 was found to be an independent prognostic marker. Lack of p27 was associated with poor prognosis (2.9 risk ratio for death; P = 0.003). The absence of p27 protein expression is thus a powerful negative prognostic marker in colorectal carcinomas, particularly in stage II tumors, and thereby may help in the selection of patients who will benefit from adjuvant therapy. These data suggest that aggressive tumors may result from the selection of a clone or clones that lack p27 due to increased proteasome-mediated degradation.

Cell cycle: will the real Cdk-activating kinase please stand up.

Activation of a cyclin-dependent kinase (Cdk) requires its association with a cyclin subunit and phosphorylation on a conserved residue by the Cdk-activating kinase (CAK). The discovery in budding yeast of a novel CAK that is structurally distinct from the CAKs of other eukaryotes raises questions about the 'true' CAK.

CDC25 phosphatases as potential human oncogenes.

Cyclin-dependent kinases (CDKs) are activated by CDC25 phosphatases, which remove inhibitory phosphate from tyrosine and threonine residues. In human cells, CDC25 proteins are encoded by a multigene family, consisting of CDC25A, CDC25B, and CDC25C. In rodent cells, human CDC25A or CDC25B but not CDC25C phosphatases cooperate with either Ha-RASG12V or loss of RB1 in oncogenic focus formation. Such transformants were highly aneuploid, grew in soft agar, and formed high-grade tumors in nude mice. Overexpression of CDC25B was detected in 32 percent of human primary breast cancers tested. The CDC25 phosphatases may contribute to the development of human cancer.

Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.

The p27 mammalian cell cycle protein is an inhibitor of cyclin-dependent kinases. Both in vivo and in vitro, p27 was found to be degraded by the ubiquitin-proteasome pathway. The human ubiquitin-conjugating enzymes Ubc2 and Ubc3 were specifically involved in the ubiquitination of p27. Compared with proliferating cells, quiescent cells exhibited a smaller amount of p27 ubiquitinating activity, which accounted for the marked increase of p27 half-life measured in these cells. Thus, the abundance of p27 in cells is regulated by degradation. The specific proteolysis of p27 may represent a mechanism for regulating the activity of cyclin-dependent kinases.

Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP).

The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.

p13suc1 of Schizosaccharomyces pombe regulates two distinct forms of the mitotic cdc2 kinase.

suc1 is an essential gene initially identified for its ability to rescue certain temperature-sensitive alleles of cdc2 in Schizosaccharomyces pombe. The role of suc1 in the regulation of the cdc2 kinase is not well understood. In our study, we have characterized the biochemical effect of loss of suc1 function on specific cdc2-cyclin complexes. We show that the cig1 cyclin is associated with cdc2 and that the cdc2-cig1 kinase is activated at mitosis, with kinetics similar to those of the cdc2-cdc13 kinase. We provide evidence that loss of suc1 function affects the kinase activity of the two distinct mitotic forms of the cdc2 kinase. We also show that a dramatic increase in the level of the cdc13 protein is associated with loss of suc1. These results suggest that mitosis cannot be properly completed in the absence of suc1, possibly because of an increase in the level of cdc2-cdc13 complex, and support the idea of a role for suc1 in the regulation of multiple forms of the cdc2 kinase.

Mammalian G1 cyclins.

D- and E-type cyclins regulate the progression of mammalian cells through the G1 phase of the cell cycle. The mechanisms responsible for the accumulation and activation of kinases dependent on cyclins D and E in both normal and cancerous cells have recently been uncovered. Overexpression of cyclin D1 protein as a consequence of genetic rearrangements, and deletions or mutations of the p16INK4 gene have been demonstrated in a large variety of human cancers, including breast and esophageal carcinomas, lymphomas, bladder carcinoma, pancreatic adenocarcinoma and familial melanoma.

Molecular cloning of PISSLRE, a novel putative member of the cdk family of protein serine/threonine kinases.

Several members of the cdk family of protein kinases are involved in the regulation of the eukaryotic cell cycle. Using a PCR-based strategy we have screened different human tumor cell lines for cdk-related cDNAs. One clone isolated from the bladder carcinoma cell line RT112 encodes a novel protein kinase named PISSLRE, based on its predicted sequence at the conserved PSTAIRE motif. PISSLRE showed 50% amino acid identity with the previously isolated p58KGTA. PISSLRE contained all the structural elements featured by cyclin dependent kinases, including a proline in the PSTAIRE motif, which might be important for cyclin binding. PISSLRE was found expressed as 2.0 kb messenger RNA in a variety of human cell lines. Its expression was not restricted to tumor cells as it was detectable also in normal fibroblasts. In adult tissues, PISSLRE mRNA showed the highest expression in lung, liver and kidney. The broad expression pattern in adult tissues might suggest that PISSLRE could be involved in processes distinct from cell proliferation.